Nitric oxide (NO) is responsible for nitrergic neurotransmission in the gut and its release is dependent on its de novo synthesis by nNOS (neuronal nitric oxide synthase). The magnitude of NO synthesis and release during neurotransmission may be related to the fraction of catalytically active nNOS out of a larger pool of inactive nNOS in the nerve terminals. The purpose of the current study was to identify catalytically active and inactive pools of nNOS in the varicosities from mice gut. Enteric varicosities were confirmed as nitrergic by colocalization of nNOS with the nerve varicosity marker synaptophysin. Low temperature SDS PAGE of these varicosity extracts showed 320kD, 250kD and 155kD bands when blotted with anti-nNOS_1422-1433 and 320 and 155kD bands when blotted with anti-nNOS_1-20 antibodies respectively. The 320kD and 155kD bands represent dimers and monomers of nNOS; the 250kD and 135kD bands represent dimers and monomers of nNOS. Immunoprecipitation with calmodulin (CaM) showed that a portion of nNOS dimer was bound with CaM. On the other hand, a portion of nNOS dimer, nNOS dimer and all monomers lacked CaM binding. The CaM-lacking nNOS fractions reacted with anti-serine847-phospho-nNOS. In vitro assays of NO production revealed that only the CaM-bound dimeric nNOS was catalytically active; all other forms were inactive. We suggest that the amount of catalytically active nNOS dimers may be regulated by serine847 phosphorylation and equilibrium between dimers and monomers of nNOS.